[5-Carboxamido or 5-fluoro]-[2′,3′-unsaturated or 3′-modified]-pyrimidine nucleosides

ABSTRACT

A method and composition for the treatment of HIV and HBV infections in humans and other host animals is disclosed that includes the administration of an effective amount of a [5-carboxamido or 5-fluoro]-2′,3′-dideoxy-2′,3′-didehydro-pyrimidine nucleoside or a [5-carboxamido or 5-fluoro]-3′-modified-pyrimidine nucleoside, or a mixture or a pharmaceutically acceptable derivative thereof, including a 5′ or N 4  alkylated or acylated derivative, or a pharmaceutically acceptable salt thereof, in a pharmaceutically acceptable carrier.

This application is a continuation of U.S. Ser. No. 09/310,823, filed onMay 12, 1999, now U.S. Pat. No. 6,232,300, which is a continuation ofU.S. Ser. No. 09/001,084 filed on Dec. 30, 1997, now U.S. Pat. No.5,905,070, which is a continuation of U.S. Ser. No. 08/379,276, filed onJan. 27, 1995, now U.S. Pat. No. 5,703,058.

BACKGROUND OF THE INVENTION

This invention is in the area of biologically active nucleosides, andspecifically includes antiviral compositions that include a[5-carboxamido or 5-fluoro]-2′, 3′-dideoxy-2′,3′-didehydro-pyrimidinenucleoside or [5-carboxamido or 5-fluoro]-3′-modified-pyrimidinenucleoside, or its physiologically acceptable derivative, orphysiologically acceptable salt.

In 1981, acquired immune deficiency syndrome (AIDS) was identified as adisease that severely compromises the human immune system, and thatalmost without exception leads to death. In 1983, the etiological causeof AIDS was determined to be the human immunodeficiency virus (HIV). TheWorld Health Organization estimates that currently 13 million peopleworldwide are infected with HIV and that forty million people will beinfected by the year 2000. Each day approximately 5,000 people are newlyinfected.

In 1985, it was reported that the synthetic nucleoside3′-azido-3′-deoxythymidine (AZT) inhibits the replication of a humanimmunodeficiency virus. Since then, a number of other syntheticnucleosides, including 2′,3′dideoxyinosine (DDI), 2′,3′-dideoxycytidine(DDC), and 2′,3′-dideoxy-2′,3′-didehydrothymidine (D4T), have beenproven to be effective against HIV. After cellular phosphorylation tothe 5′-triphosphate by cellular kinases, these synthetic nucleosides areincorporated into a growing strand of viral DNA, causing chaintermination due to the absence of the 3′-hydroxyl group. They can alsoinhibit the viral enxyme reverse transcriptase.

The success of various synthetic nucleosides in inhibiting thereplication of HIV in vivo or in vitro has led a number of researchersto design and test nucleosides that substitute a heteroatom for thecarbon atom at the 3′-position of the nucleoside. Norbeck, et al.,disclosed that (±)-1-[(2β,4β)-2-(hydroxymethyl)-4-dioxolanyl]thymine(referred to as (±)-dioxolane-T) exhibits a modest activity against HIV(EC₅₀ of 20 μM in ATH8 cells), and is not toxic to uninfected controlcells at a concentration of 200 μM. Tetrahedron Letters 30 (46), 6246,(1989). European Patent Application Publication No. 0 337 713 and U.S.Pat. No. 5,041,449, assigned to IAF BioChem International, Inc.,disclose that racemic 2-substituted-4-substituted-1,3-dioxolanes exhibitantiviral activity.

U.S. Pat. No. 5,047,407 and European Patent Application Publication No.O 382 526, also assigned to IAF Biochem International, Inc. disclose anumber of racemic 2-substituted-5-substituted-1,3-oxathiolanenucleosides with antiviral activity, and specifically report that theracemic mixture (about the C4′-position) of the C1′-β isomer of2-hydroxymethyl-5-(cytosin-1-yl)-1,3-oxathiolane (referred to below as(±)-BCH-189) has approximately the same activity against HIV as AZT, andno cellular toxicity at the tested levels. (±)-BCH-189 has also beenfound to inhibit the replication of AZT-resistant HIV isolates in vitrofrom patients who have been treated with AZT for longer than 36 weeks.The (−)-enantiomer of the β-isomer of BCH-189, known as 3TC, which ishighly potent against HIV and exhibits little toxicity, is in the finalstages of clinical review for the treatment of HIV.

It has also been disclosed that(−)-cis-2-hydroxymethyl-5-(5-fluorocytosin-1-yl)-1,3-oxathiolane (“FTC”)has potent HIV activity. Schinazi, et al., “Selective Inhibition ofHuman Imnunodeficiency Viruses by Racemates and Enantiomers ofcis-5-Fluoro-1-[2-(Hydroxymethyl)-1,3-Oxathiolane-5-yl]Cytosine”Antimicrobial Agents and Chemotherapy, November 1992, page 2423-2431.

Another virus that causes a serious human health problem is thehepatitis B virus (referred to below as “HBV”). HBV is second only totobacco as a cause of human cancer. The mechanism by which HBV inducescancer is unknown, although it is postulated that it may directlytrigger tumor development, or indirectly trigger tumor developmentthrough chronic inflammation, cirrhosis, and cell regenerationassociated with the infection.

After a two to six month incubation period in which the host is unawareof the infection, HBV infection can lead to acute hepatitis and liverdamage, that causes abdominal pain, jaundice, and elevated blood levelsof certain enzymes. HBV can cause fulminant hepatitis, a rapidlyprogressive, often fatal form of the disease in which massive sectionsof the liver are destroyed.

Patients typically recover from acute hepatitis. In some patients,however, high levels of viral antigen persist in the blood for anextended, or indefinite, period, causing a chronic infection. Chronicinfections can lead to chronic persistent hepatitis. Patients infectedwith chronic persistent HBV are most common in developing countries. Bymid-1991, there were approximately 225 million chronic carriers of HBVin Asia alone, and worldwide, almost 300 million carriers. Chronicpersistent hepatitis can cause fatigue, cirrhosis of the liver, andhepatocellular carcinoma, a primary liver cancer.

In western industrialized countries, high risk groups for HBV infectioninclude those in contact with HBV carriers or their blood samples. Theepidemiology of HBV is very similar to that of acquired immunedeficiency syndrome, which accounts for why HBV infection is commonamong patients with AIDS or AIDS-related complex. However, HBV is morecontagious than HIV.

Both FTC and 3TC exhibit activity against HBV. Furman, et al., “TheAnti-Hepatitis B Virus Activities, Cytotoxicities, and Anabolic Profilesof the (−) and (+) Enantiomers ofcis-5-Fluoro-1-[2-(Hydroxymethyl)-1,3-oxathiolane-5-yl]Cytosine”Antimicrobial Agents and Chemotherapy, December 1992, page 2686-2692.

A human serum-derived vaccine has been developed to immunize patientsagainst HBV. While it has been found effective, production of thevaccine is troublesome because the supply of human serum from chroniccarriers is limited, and the purification procedure is long andexpensive. Further, each batch of vaccine prepared from different serummust be tested in chimpanzees to ensure safety. Vaccines have also beenproduced through genetic engineering. Daily treatments withα-interferon, a genetically engineered protein, has also shown promise.

In light of the fact that acquired immune deficiency syndrome,AIDS-related complex, and hepatitis B virus have reached epidemic levelsworldwide, and have tragic effects on the infected patient, thereremains a strong need to provide new effective pharmaceutical agents totreat these diseases and that have low toxicity to the host.

Therefore, it is an object of the present invention to provide a methodand composition for the treatment of human patients infected with HIV.

It is another object of the present invention to provide a method andcomposition for the treatment of human patients or other host animalsinfected with HBV.

SUMMARY OF THE INVENTION

A method and composition for the treatment of HIV and HBV infections inhumans and other host animals is disclosed that includes theadministration of an effective amount of a [5-carboxamido or5-fluoro]-2′,3′-dideoxy-2′, 3′-didehydro-pyrimidine nucleoside or a[5-carboxamido or 5-fluoro]-3′-modified-pyrimidine nucleoside, or amixture or a pharmaceutically acceptable derivative thereof, including a5′ or N⁴ alkylated or acylated derivative, or a pharmaceuticallyacceptable salt thereof, in a pharmaceutically acceptable carrier.

Specifically, compounds of the structure:

wherein:

X is O, S, CH₂, CHF, or CF₂;

Y is O, S, CH₂, CHF, CF₂;

Z is independently O, S or Se;

R₁ is independently H or F;

R₂ is independently H, OH, C₁ to C₆ alkyl, or C(O)(C₁ to C₆ alkyl);

R₃ is H, C(O)(C₁-C₆ alkyl); alkyl, or mono-, di- or triphosphate; and

R₄ is independently H, F, Cl, Br, I, OH,

—O(C₁-C₆alkyl), —SH, —S(C₁-C₆alkyl); or

—C₁-C₆alkyl.

In a preferred embodiment for 2′,3′-dideoxy-2′,3′-didehydro-nucleosides,Y is O or S; Z is O; R₁ is H; R is H; and R₃ is H. In a preferredembodiment for the 3′-modified lipyrimidine nucleosides, X is O or S; Yis O; Z is O; R₁ is H; R₂ is H; R₃ is H, and R₄ is independently H or F.The term “independently” means that the groups can vary within thecompound.

Preferred compounds include the racemic mixture, β-D and β-L isomers ofthe following compounds:2-hydroxymethyl-5(5′-carboxamidouracil-1′-yl)-1,3-oxathiolane;2-hydroxymethyl-4-(N-5′-carboxamidouracil-1′-yl)-1,3-dioxolane;2-hydroxymethyl-4-(N-5′-flubrocytosin-1′-yl)-1,3-dithiolane;2-hydroxymethyl-4-(N-5′-carboxamidouracil-1′-yl)-1,3-dithiolane;2-hydroxymethyl-4-(N-5′-fluorocytosin-1′-yl)-1,3-oxathiolane;2-hydroxymethyl-4-(N-5′-carboxamidouracil-1′-yl)-1,3-oxathiolane;2′,3′-dideoxy-2′,3′-didehydro-5-fluorocytidine;2′,3′-dideoxy-2′,3′-didehydro-5-carboxamidocytidine;2′,3′-dideoxy-5-fluorocytidine; 2′,3′-dideoxy-5-carboxamidocytidine;2′,3′-dideoxy-2′,3′-didehydro-2′, 5-difluorocytidine;2′,3′-dideoxy-2′,3′-didehydro-2′-fluoro-5-carboxamidocytidine,2′,3′-dideoxy-2′,3′-didehydro-3′, 5-difluorocytidine;2′,3′-dideoxy-2′,3′-didehydro-3′-fluoro-5-carboxamidocytidine;2′,3′-dideoxy-2′,3′-didehydro-2′,3′, 5-trifluorocytidine;2′,3′-dideoxy-2′,3′-didehydro-2′, 3′-difluoro-5-carboxamidocytidine;2′,3′-dideoxy-2′,3′-didehydro-5-fluorocytidine;2′,3′-dideoxy-2′,3′-didehydro-5-carboxamidocytidine;2′,3′-dideoxy-5-fluorocytidine; 2′,3′-dideoxy-5-carboxamidocytidine;2′,3′-dideoxy-2′,3′-didehydro-2′,5-difluorocytidine;2′,3′-dideoxy-2′,3′-didehydro-2′-fluoro-5-carboxamidocytidine;2′,3′-dideoxy-2′,3′-didehydro-3′,5-difluorouridine;2′,3′-dideoxy-2′,3′-didehydro-3′-fluoro-5-carboxamidouridine;2′,3′-dideoxy-2′,3′-didehydro-2′,3′,5-trifluorouridine; and2′,3′-dideoxy-2′,3′-didehydro-2′,3′-difluoro-5-carboxamidouridine.

In another embodiment, the active compound or its derivative or salt canbe administered in combination or it alternation with another antiviralagent, such as an anti-HIV agent or anti-HBV agent, including thosedescribed above. In general, during alternation therapy, an effectivedosage of each agent is administered serially, whereas in combinationtherapy, an effective dosage of two or more agents are administeredtogether. The dosages will depend on absorption, inactivation, andexcretion rates of the drug as well as other factors known to those ofskill in the art. It is to be noted that dosage values will also varywith the severity of the condition to be alleviated. It is to be furtherunderstood that for any particular subject, specific dosage regimens andschedules should be adjusted over time according to the individual needand the professional judgment of the person administering or supervisingthe administration of the compositions.

Nonlimiting examples of antiviral agents that can be used in combinationwith the compounds disclosed herein include the (−)-enantiomer of2-hydroxymethyl-5-(5-fluorocytosin-1-yl)-1,3-oxathiolane (FTC); the(−)-enantiomer of 2-hydroxymethyl-5-(cytosin-1-yl)-1,3-oxathiolane(3TC); carbovir, acyclovir, interferon, famciclovir, penciclovir, AZT,DDI, DDC, L-(−)-FMAU, and D4T.

The compounds can also be used to treat equine infectious anemia virus(EIAV), feline immunodeficiency virus, and simian imunodeficiency virus.(Wang, S., Montelaro, R., Schinazi, R. F., Jagerski, B., and Mellors, J.W.: Activity of nucleoside and non-nucleoside reverse transcriptseinhibitors (NNRTI) against equine infectious anemia virus (EIAV). FirstNational Conference on Human Retroviruses and Related Infections,Washington, D.C., Dec. 12-16, 1993; Sellon D. C., Equine InfectiousAnemia, Vet. Clin. North Am. Equine Pract. United States, 9: 321-336,1993; Philpott, M. S., Ebner, J. P., Hoover, E. A., Evaluation of9-(2-phosphonylmethoxyethyl) adenine therapy for feline immunodeficiencyvirus using a quantative polymerase chain reaction, Vet. Immunol.Immunopathol. 35:155-166, 1992.)

DETAILED DESCRIPTION OF THE INVENTION

As used herein, the term “enantiomericaily enriched nucleoside” refersto a nucleoside composition that includes at least 95% to 98%, or morepreferably, 99% to 100%, of a single enantiomer of that nucleoside.

The term C₁-C₆ alkyl includes methyl, ethyl, propyl, isopropyl, butyl,isobutyl, t-butyl, pentyl, cyclopehtyl, isopentyl, neopentyl, hexyl,isohexyl, cyclohexyl, cyclohexylmethyl, 3-methylpentyl,2,2-dimethylbutyl, and 2,3-dimethylbutyl.

The invention as disclosed herein is a method and composition for thetreatment of HIV and HBV infections, and other viruses replicating inlike manner, in humans or other host animals, that includesadministering an effective amount of a [5-carboxamido or5-fluoro]-2′,3′-dideoxy-2′,3′-didehydro-pyrimidine nucleoside or[5-carboxamido or 5-fluoro]-3′-modified-pyrimidine nucleoside, apharmaceutically acceptable derivative, including a 5′ or, N⁴ alkylatedor acylated derivative, or a pharmaceutically acceptable salt thereof,in a pharmaceutically acceptable carrier. The compounds of thisinvention either possess antiretroviral activity, such as anti-HIV-1,anti-HIV-2, anti-HBV, and anti-simian immunodeficiency virus (anti-SIV)activity themselves or are metabolized to a compound that exhibitsantiretroviral activity.

The disclosed compounds or their pharmaceutically acceptable derivativesor salts or pharmaceutically acceptable formulations containing thesecompounds are useful in the prevention and treatment of HIV infectionsand other related conditions such as AIDS-related complex (ARC),persistent generalized lymphadenopathy (PGL), AIDS-related neurologicalconditions, anti-HIV antibody positive and HIV-positive conditions,Kaposi's sarcoma, thrombocytopenia purpurea and opportunisticinfections. In addition, these compounds or formulations can be usedprophylactically to prevent or retard the progression of clinicalillness in individuals who are anti-HIV antibody or HIV-antigen positiveor who have been exposed to HIV.

The compound and its pharmaceutically acceptable derivatives orpharmaceutically acceptable formulations containing the compound or itsderivatives are also useful in the prevention and treatment of HBVinfections and other related conditions such as anti-HBV antibodypositive and HBV-positive conditions, chronic liver inflammation causedby HBV, cirrhosis, acute hepatitis, fulminant hepatitis, chronicpersistant hepatitis, and fatigue. These compounds or formulations canalso be used prophylactically to prevent or retard the progression ofclinical illness in individuals who are anti-HBV antibody or HBV-antigenpositive or who have been exposed to HBV.

The compound can be converted into a pharmaceutically acceptable esterby reaction with an appropriate esterifying agent, for example, an acidhalide or anhydride. The compound or its pharmaceutically acceptablederivative can be converted into a pharmaceutically acceptable saltthereof in a conventional manner, for example, by treatment with anappropriate base. The ester or salt of the compound can be convertedinto the parent compound, for example, by hydrolysis.

In summary, the present invention includes the following features:

(a) [5-carboxamido or 5-fluoro]-2′,3′-dideoxy-2′,3′-didehydro-pyrimidinenucleosides and [5-carboxamido or 5-fluoro]-3′ -modified-pyrimidinenucleosides, as outlined above, and pharmaceutically acceptablederivatives and salts thereof;

(b) [5-carboxamido or 5-fluoro]-2′,3′-dideoxy-2′,3′-didehydro-pyrimidinenucleosides and [5-carboxamido or 5-fluoro]-3′-modified-pyrimidinenucleosides, and pharmaceutically acceptable derivatives and saltsthereof for use in medical therapy, for example for the treatment orprophylaxis of a HIV or HBV infection;

(c) use of [5-carboxamido or5-fluoro]-2′,3′-dideoxy-2′,3′-didehydro-pyrimidine nucleosides and[5-carboxamido or 5-fluoro]-3′-modified-pyrimidine nucleosides, andpharmaceutically acceptable derivatives and salts thereof in themanufacture of a medicament for treatment of a HIV or HBV infection;

(d) pharmaceutical formulations comprising [5-carboxamido or5-fluoro]-2′,3′-dideoxy-2′,3′-didehydro-pyrimidine nucleosides and[5-carboxamido or 5-fluoro]-3′-modified-pyrimidine nucleosides or apharmaceutically acceptable derivative or salt thereof together with apharmaceutically acceptable carrier or diluent; and

(e) processes for the preparation of [5-carboxamido or5-fluoro]-2′,3′-dideoxy-2′,3′-didehydro-pyrimidine nucleosides and[5-carboxamido or 5-fluoro]-3′-modified-pyrimidine nucleosides, asdescribed in more detail below.

I. Active Compound, and Physiologically Acceptable Derivatives and SaltsThereof

The antivirally active compounds disclosed herein are [5-carboxamido or5-fluoro]-2′,3′-dideoxy-2′,3′-didehydro-pyrimidine nucleosides and[5-carboxamido or 5-fluoro]-3′-modified-pyrimidine nucleosides, in theracemic or β-D or β-L enantiomerically enriched form.

The active compound can be administered as any derivative that uponadministration to the recipient, is capable of providing directly orindirectly, the parent compound, or that exhibits activity itself.Nonlimiting examples are the pharmaceutically acceptable salts(alternatively referred to as “physiologically acceptable salts”), andthe 5′ and N⁴ acylated or alkylated derivatives of the active compound(alternatively referred to as “physiologically active derivatives”). Inone embodiment, the acyl group is a carboxylic acid ester in which thenon-carbonyl moiety of the ester group is selected from straight,branched, or cyclic alkyl, alkoxyalkyl including methoxymethyl, aralkylincluding benzyl, aryloxyalkyl such as phenoxymethyl, aryl includingphenyl optionally substituted with halogen, C₁ to C₄ alkyl or C₁ to C₄alkoxy, sulfonate esters such as alkyl or aralkyl sulphonyl includingmethanesulfonyl, the mono, di or triphosphate ester, trityl ormonomethoxytrityl, substituted benzyl, trialkylsilyl (e.g.dimethyl-t-butylsilyl) or diphenylmethylsilyl. Aryl groups in the estersoptimally comprise a phenyl group. The alkyl group can be straight,branched, or cyclic, and is optimally a C₁ to C₁₈ group.

Modifications of the active compound, specifically at the N⁴ and 5′-Opositions, can affect the bioavailability and rate of metabolism of theactive species, thus providing control over the delivery of the activespecies. Further, the modifications can affect the antiviral activity ofthe compound, in some cases increasing the activity over the parentcompound. This can easily be assessed by preparing the derivative andtesting its antiviral activity according to the methods describedherein, or other method known to those skilled in the art.

Since the 1′ and 4′ carbons of the carbohydrate of the nucleoside(referred to below generically as the sugar moiety) of the nucleosidesare chiral, their nonhydrogen substituents (the pyrimidine or purinebase and the CH₂OR groups, respectively) can be either cis (on the sameside) or trans (on opposite sides) with respect to the sugar ringsystem. The four optical isomers therefore are represented by thefollowing configurations (when orienting the sugar moiety in ahorizontal plane such that the Y substituent is in the back): cis (withboth groups “up”, which corresponds to the configuration of naturallyoccurring nucleosides), cis (with both groups “down”, which is anonnaturally occurring configuration), trans (with the C2′ substituent“up” and the C4′ substituent “down”), and trans (with the C2′substituent “down” and the C4′ substituent “up”). The “D-nucleosides”are cis nucleosides in a natural configuration and the “L-nucleosides”are cis nucleosides in the nonnaturally occurring configuration.

As known to those skilled in the art of nucleoside chemistry, in somecases, one of the β-cis enantiomers can be more active, or less toxic,than the other enantiomer. This can be easily determined by separatingthe enantiomers and testing the activity and cytotoxicity using standardassays.

II. Preparation of the Active Compounds

The nucleosides disclosed herein for the treatment of HIV and HBVinfections in a host organism can be prepared according to publishedmethods. β-L-Nucleosides can be prepared from methods disclosed in, orstandard modifications of methods disclosed in, for example, thefollowing publications: Jeong, et al., J. of Med. Chem., 36, 182-195,1993; European Patent Application Publication No. 0 285 884;Génu-Dellac, C., G. Gosselin, A.-M. Aubertin, G. Obert, A. Kirn, andJ.-L. Imbach, 3-Substituted thymine α-L-nucleoside derivatives aspotential antiviral agents; synthesis and biological evaluation,Antiviral Chem. Chemother. 2:83-92 (1991); Johansson, K. N. G., B. G.Lindborg, and R. Noreen, European Patent Application 352 248; Mansuri,M. M., V. Farina, J. E. Starrett, D. A. Benigni, V. Brankovan, and J. C.Martin, Preparation of the geometric isomers of DDC, DDA, D4C and D4T aspotential anti-HIV agents, Bioorg. Med. Chem. Lett. 1:65-68 (1991);Fujimori, S., N. Iwanami, Y. Hashimoto, and K. Shudo, A convenient andstereoselective synthesis of 2′-deoxy-β-L-ribonucleosides, Nucleosides &Nucleotides 11:141-349 (1992); Génu-Dellac, C., G. Gosselin, A.-M.Aubertin, G. Obert, A. Kirn, and J.-L. Imbach, 3-Substituted thymineα-L-nucleoside derivatives as potential antiviral agents; synthesis andbiological evaluation, Antiviral Chem. Chemother. 2:83-92 (1991); Holy,A, Synthesis of 2′-deoxy-L-uridine, Tetrahedron Lett. 2:189-192 (1992);Holy, A., Nucleic acid components and their analogs. CLIII. Preparationof 2′-deoxy-L-ribonucleosides of the pyrimidine series. Collect CzechChem Commun. 37:4072-4087 (1992); Holy, A, 2′-deoxy-L-uridine: Totalsynthesis of a uracil 2′-deoxynucleoside from a sugar 2-aminooxazolinethrough a 2,2′ anhydronucleoside intermediate. In: Townsend L B, TipsonR S, ed. Nucleic Acid Chem, New York: Wiley, 1992: 347-353. vol 1)(1992); Okabe, M., R.-C. Sun, S. Tan, L. Todaro, and D. L. Coffen,Synthesis of the dideoxynucleosides ddC and CNT from glutamic acid,ribonolactone, and pyrimidine bases. J Org Chem. 53:4780-4786 (1988);Robins, M. J., T. A. Khwja, and R. K. Robins. Purine nucleosides. XXIX.Synthesis of 21-deoxy-L-adenosine and 21-deoxy-L-guanosine and theiralpha anomers. J Org Chem. 35:363-639 (1992); Génu-Dellac, C., GosselinG., Aubertin A-M, Obert G., Kirn A., and Imbach J-L, 3′-Substitutedthymine α-L-nucleoside derivatives as potential antiviral agents;synthesis and biological evaluation. Antiviral Chem. Chemother.2(2):83-92 (1991); Génu-Dellac, C., Gosselin G., Imbach J-L; Synthesisof new 2′-deoxy-3′-substituted-α-L-threo-pentofuranonucleosides ofthymine as a potential antiviral agents. Tet Lett 32(1):79-82 (1991);Génu-Dellac, C., Gosselin G., Imbach J-L, Preparation of new acylatedderivatives of L-arabino-furanose and 2-deoxy-1-erythro-pentofuranose asprecursors for the synthesis of 1-pentofuranosyl nucleosides.216:240-255 (1991); and Génu-Dellac, C., Gosselin G., Puech F, et al.Systematic synthesis and antiviral evaluation of α-L-arabinofuranosyland 2′-deoxy-α-L-erythro-pento-furanosyl nucleosides of the fivenaturally occurring nucleic acid bases. 10(b):1345-1376 (1991).

β-D-Dioxolane-nucleosides can be prepared as disclosed in detail inPCT/US91/09124. The process involves the initial preparation of (2R,4R)-and (2R,4S)-4-acetoxy-2-(protected-oxymethyl)-dioxolane from1,6-anhydromannose, a sugar that contains all of the necessarystereochemistry for the enantiomerically pure final product, includingthe correct diastereomeric configuration about the 1 position of thesugar (that becomes the 4′-position in the later formed nucleoside). The(2R,4R)- and (2R,4S)-4-acetoxy-2-(protected-oxymethyl)-dioxolane iscondensed with a desired heterocyclic base in the presence of SnCl₄,other Lewis acid, or trimethylsilyl triflate in an organic solvent suchas dichloroethane, acetonitrile, or methylene chloride, to provide thestereochemically pure dioxolane-nucleoside.

Enzymatic methods for the separation of D and L enantiomr ofcis-nucleoside are disclosed in, for example, Nucleosides andNucleotides, 12(2), 225-236 (1993); European Patent Application Nos.92304551.2 and 92304552.0 filed by Biochem Pharma, Inc.; and PCTPublication Nos. WO 91/11186, WO 92/14729, and WO 92/14743 filed byEmory University.

Separation of the acylated or alkylated racemic mixture of D and Lenantiomers of cis-nucleosides can be accomplished by high performanceliquid chromatography with selected chiral stationary phases, asdisclosed, for example, in PCT Publication No. WO 92/14729.

Mono, di, and triphosphate derivatives of the active nucleosides can beprepared as described according to published methods, The monophosphatecan be prepared according to the procedure of Imai et al., J. Org.Chem.,34(6), 1547-1550 (June 1969). The diphosphate can be preparedaccording to the procedure of Davisson et al., J. Org. Chem., 52(9),1794-1801 (1987). The triphosphate can be prepared according to theprocedure of Hoard et al., J. Am. Chem. Soc., 87(8), 1785-1788 (1965).

Other references disclosing useful methods that can be used or adaptedfor the preparation of the active compounds include Hutchinson, D. W.“New Approaches to the Synthesis of Antiviral Nucleosides” TIBTECH,1990, 8, 348; Agrofoglio, L. et al. “Synthesis of CarbocyclicNucleosides” Tetrahedron, 1994, 50, 10611; Dueholm, K. L.; Pederson, E.B. Synthesis, 1994, 1; Wilson, L. J., Choi, W.-B., Spurling, T.,Schinazi, R. F., Cannon, D., Painter, G. R., St.Clair, M., and Furman,P. A. The Synthesis and Anti-HIV Activity of Pyrimidine DioxanylNucleoside Analogues. Bio. Med. Chem. Lett., 1993, 3, 169-174; Hoong, L.K., Strange, L. E., Liotta, D. C., Koszalka, G. W., Burns, C. L.,Schinazi, R. F. Enzyme-mediated enantioselective preparation of theantiviral agent 2′, 3′-dideoxy-5-fluoro-3′-thiacytidine [(−)-FTC] andrelated compounds. J. Org. Chem., 1992, 57, 5563-5565; Choi, W.-B.,Wilson, L. J., Yeola, S., Liotta, D. C., Schinazi, F. R. In situcomplexation directs the stereochemistry of N-glycosylation in thesynthesis of oxathiolanyl and dioxolanyl nucleoside analogues. J. Amer.Chem. Soc., 1991, 113, 9377-9379; Choi, W.-B., Yeola, S., Liotta, D. C.,Schinazi, R. F., Painter, G. R., Davis, M., St.Clair, M., Furman, P. A.The Synthesis, Anti-HIV and Anti-HBV Activity of Pyrimidine OxathiolaneNucleoside Analogues. Bio. Med. Chem. Lett., 1993, 3, 693-696; Wilson,J. E., Martin, J. L., Borrota-Esoda, K., Hopkins, S. E., Painter, G. R.,Liotta, D. C., Furman, P. A. The 5′-Triphosphates of the (−)- and(+)-Enantiomers of Cis-5-Fluoro-1-[2-(hydroxymethyl)-1,3-Oxathioan-5-yl]Cytosine Equally Inhibit Human Immunodeficiency Virus Type-1 ReverseTranscriptase. Antimicrob. Agents Chemother., 1993, 37, 1720-1722.

The following working example provides a method for the preparation of5-carboxamide-2′,3′-dideoxy-3′-thiauridine. Melting points weredetermined on an Electrothermal IA 8100 digital melting point apparatusand are uncorrected. ¹H and ¹³C NMR spectra were recorded on a GeneralElectric QE-300 (300 MHz) spectrometer; chemical shifts are reported inparts per million (d) and signals are quoted as s (singlet), d(doublet), t (triplet), or m (multiplet). UV spectrum were recorded onShimadzu UV-2101PC spectrophotometer and FTIR spectra were measured on aNicolet Impact 400 spectrometer. Mass spectroscopy was performed withJEOL (JMS-SX102/SX102A/E) spectrometer. Experiments were monitored usingTLC analysis performed on Kodak chromatogram sheets precoated withsilica gel and a fluorescent indicator. Column chromatography, employingsilica gel (60-200 mesh; Fisher Scientific, Fair Lawn, N.J.) was usedfor the purification of products. Tetrakis-(triphenylphosphine)palladium(0) and other chemicals were purchased from Aldrich Chemical Company(Milwaukee, Wis.). Microanalyses were performed at Atlantic MicrolabInc. (Norcross, Ga.). NMR Enzymes were purchased from AmanoInternational Enzyme Co. (Troy, Va.).

EXAMPLE 1 Preparation of 5-Carbonimide-2′,3 -dideoxy-3′-thiauridine

Coupling of 1-O-acetyl-5′-butyryl-3-thiafuranose with 5 -iodo-cytidineusing tin chloride afforded the protected b-isomer of5′-butyryl-2′,3′-deoxy-5-iodo-3′-thia-cytidine with goodstereoselectivity.

To a solution of 5′-butyryl-2′,3′-deoxy-5-iodo-3′-thiacytidine (1.63 g;3.83 mmol) in 100 ml of anhydrous MeOH was addedtetrakis-(triphenylphosphine)palladium (0) (0.16 g, 0.14 mmol) and Et₃N(0.8 ml). The reaction mixture was maintained under a CO atmosphere for6 h while heating at 40° C. The solution was concentrated to dryness invacuo, dissolved in CH₂Cl₁ then filtered. The resultant precipitate wasdissolved in hot CHCl₃ to give after crystallization the desired product5-carboxylic acid methyl ester-2′,3′-dideoxy-3′-thiacytidine (0.7 g,62%) as a white solid. m.p. 217-221°C.; ¹H NMR (DMSO) d 3.2-3.3 (m, 2H,H-2′ and H-2″), 3.75 (s, 3H, OCH₃), 3.8-4.0 (m, 2H H-5′ and H-5″), 5.36(m, 1H, OH-5′), 5.49 (t, 1H, H-4′, J_(4′,5′)=4.0, 6.21 (m, 1H, H-1′),7.7 and 8.1 (2 br s, 1H each, NH₂), 9.0 (s, 1H, H-6); m/z (LSIMS) 288(M+H)⁺; Anal. (C₁₀H₁₃N₃O₅S) C, H, N, S.

To a solution of 5-carboxylic acid methyl ester-2′,3′-dideoxy-3′-thiacytidine (0.2 g, 0.69 mmol) in anhydrous MeOH was added(50 ml) a 2 M solution at of NH₃-MeOH and a catalytic amount of NaCN (20mg). The resulting solution was stirred at 100 degrees for 20 h and thenconcentrated in vacuo. The residue was chromatographed on silica gelusing CH₂Cl₂/MeOH (90:10) as eluent to give 5-carboxylic acidamide-2′,3′-dideoxy-3′-thiacytidine (0.12 g, 63%) as a white solid. m.p.190-192 degrees; ¹H NMR (DMSO) d 3.18 (dd, 1H, H-2′ or H-2″,J_(2′,2″)=10.2, J_(2′or 2″,1′)=1.4), 3.41 (dd, 1H, H-2′ or H-2″,J_(2′,2″)=10.1, J_(2′or 2″,1′)=1.5), 3.8-4.0 (m, 2H , H-5′ and H-5″),5.36 (t, 1H, H-4′, J_(4′,5′)=4.0), 5.5 (br s, 1H, OH-5′), 6.21 (dd, 1H,H-1′, J_(1′,2′or 2″)=4.3, J_(1′,2′or 2″)=1.9), 7.5 (br s, 2H, NH2), 7.8and 8.4 (2 br s, 1H each, NH₂), 8.6 (s, 1H, H-6); m/z (LSIMS) 273(M+H)⁺; Anal. (C₉H₁₂N₄O₄S) C, H, N, S.

EXAMPLE 2 Preparation of β-D and β-L Enantiomers of 5-Caraboxylic AcidAmide -2′,3′-dideoxy-3′-thiacytidine

5′-Butyryl-2′,3′-deoxy-5-iodo-3′-thiacytidine (3 g, 7 mmol) wasdissolved in 900 ml of 4/1 pH 8 buffer/CH₃CN. The clear solution wasstirred and treated with 1000 units of pig liver esterase (PLE-A,Amano). The progress of the reaction was monitored by HPLC. After 16hours (50% conversion), the reaction mixture was extracted with 2×600 mlof CHCl₃ and 600 ml of EtOAC. The organic extracts were combined, driedover MgSO₄, filtered, and concentrated to dryness, and then submitted tothe same pathway described in Example 1. The aqueous layer wasevaporated to dryness then protected on the 5′-position using butyrylchloride and submitted to the same reaction pathway.

EXAMPLE 3 Preparation of 2′,3′-Didehydro-2′,3′-dideoxy-pyrimidineNucleosides

Scheme 1 below provides a general process for the preparation of2′,3′-didehydro-2′,3′-dideoxy-pyrimidine nucleosides. This procedure canbe adapted for a wide variety of bases, and can be used to provideeither the β-D or the β-L isomer, as desired.

IV. Ability of [5-Carboxamido or 5-Flouro]-2′,3′-dideoxy-2′,3′-didehydro-pyrimidine Nucleoside or [5-Carboxamido or5-Flouro]-3′-modified-pyrimidine Nucleocides to Inhibit the Replicationof HIV and HBV

The ability of nucleosides to inhibit HIV can be measured by variousexperimental techniques. The technique used herein, and described indetail below, measures the inhibition of viral replication inphytohemagglutinin (PHA) stimulated human peripheral blood mononuclear(PBM) cells infected with HIV-1 (strain LAV). The amount of virusproduced is determined by measuring the virus-coded reversetranscriptase enzyme. The amount of enzyme produced is proportional tothe amount of virus produced.

EXAMPLE 4 Anti-HIV Activity of 5-Substituted Derivatives of 2′,3′-Dideoxy-3′-thyacytidine

A series of 5-substituted derivatives of 2′, 3′-thiacytidine and2′,3′-dideoxy-3′-thiauridine (see Table 1) were synthesized and testedfor anti-HIV activity.

Three-day-old phytohemagglutinin-stimulated PBM cells (10⁶ cells/ml)from hepatitis B and HIV-1 seronegative healthy donors were infectedwith HIV-1 (strain LAV) at a concentration of about 100 times the 50%tissue culture infectious dose (TICD 50) per ml and cultured in thepresence and absence of various concentrations of antiviral compounds.

Approximately one hour after infection, the medium, with the compound tobe tested (2 times the final concentration in medium) or withoutcompound, was added to the flasks (5 ml; final volume 10 ml). AZT wasused as a positive control.

The cells were exposed to the virus (about 2×10⁵ dpm/ml, as determinedby reverse transcriptase assay) and then placed in a CO₂ incubator.HIV-1 (strain LAV) was obtained from the Center for Disease Control,Atlanta, Ga. The methods used for culturing the PBM cells, harvestingthe virus and determining the reverse transcriptase activity were thosedescribed by McDougal et al. (J. Immun. Meth. 76, 171-183, 1985) andSpira et al. (J. Clin. Meth. 25, 97-99, 1987), except that fungizone wasnot included in the medium (see Schinazi, et al., Antimicrob. AgentsChemother. 32, 1784-1787 (1988); Id., 34:1061-1067 (1990)).

On day 6, the cells and supernatant were transferred to a 15 ml tube andcentrifuged at about 900 g for 10 minutes. Five ml of supernatant wereremoved and the virus was concentrated by centrifugation at 40,000 rpmfor 30 minutes (Beckman 70.1 Ti rotor). The solubilized virus pellet wasprocessed for determination of the levels of reverse transcriptase.Results are expressed in dpm/ml of sampled supernatant. Virus fromsmaller volumes of supernatant (1 ml) can also be concentrated bycentrifugation prior to solubilization and determination of reversetranscriptase levels.

The median effective (EC₅₀) concentration was determined by the medianeffect-method (Antimicrob. Agents Chemother. 30, 491-498 (1986).Briefly, the percent inhibition of virus, as determined frommeasurements of reverse transcriptase, is plotted versus the micromolarconcentration of compound. The EC₅₀ is the concentration of compound atwhich there is a 50% inhibition of viral growth.

Mitogen stimulated uninfected human PBM cells (3.8×10⁵ cells/ml) werecultured in the presence and absence of drug under similar conditions asthose used for the antiviral assay described above. The cells werecounted after 6 days using a hemacytometer and the trypan blue exclusionmethod, as described by Schinazi et al., Antimicrobial Agents andChemotherapy, 22(3), 499 (1982). The IC₅₀ is the concentration ofcompound which inhibits 50% of normal cell growth.

Table 1 provides the EC₅₀ values (concentration of nucleoside thatinhibits the replication of the virus by 50% in PBM cells, estimated 10%error factor) and IC₅₀ values (concentration of nucleoside that inhibits50% of the growth of mitogen-stimulated uninfected human PBM cells, CEMcells, and in Vero cells) of a number of the tested5-substituted-3′-thia-2′,3′-dideoxypyrimidine nucleosides. In the uracilseries none of the derivatives demonstrated any significant antiviralactivity. In contrast, in the cytosine series, the racemic 5-acetamidederivative was shown to have antiviral activity with a median effectiveconcentration of 0.77 micromolar and no toxicity up to 100 micromolar invarious cell lines. Similar results were obtained on evaluation of theanti-HBV activity. The racemic compound was resolved by an enzymemediated approach into the β-D and β-L enantiomers, as described inExample 2. Both 5-acetamide derivatives were effective inhibitors ofHIV-1 and HBV replication.

TABLE 1 Biological Evaluation of Various5-Substituted-3′-thia-2′,3′-dideoxypyrimidine Nucleosides AgainstHIV-1_(LA1), HSV-1_(f), and for Cytotoxicity in PBM, CEM, and VeroCells. Anti-HIV-1 Toxicity in Toxicity in Toxicity in Anti-HSV-1 in PBMCPBM cells CEM cells Vero cells in Vero cells Base 5-SubstituentConfiguration EC₅₀, μM IC₅₀, μM IC₅₀, μM IC₅₀, μM EC₅₀, μM^(a) U Nitro(±)-β-DL 122.2 >100 >100 >100 C Nitro (±)-β-DL 100.0 >100 >100 >100 UAmino (±)-β-DL 118.6 >100 >100 >100 C Amino (±)-β-DL 26.4 >100 >100 >100U Ethynyl (±)-β-DL 23.8 >100 >100 >100 C Ethynyl(±)-β-DL >100 >100 >100 >100 U Ethyl (±)-β-DL >100 >100 >100 >100 CEthyl (±)-β-DL 102.5 >100 >100 >100 U Cyano (±)-β-DL >100 >100 >100 ND CCyano (±)-β-DL >100 >100 >100 >100 U Methoxycarbonyl(±)-β-DL >100 >100 >100 >100 >100 C Methoxycarbonyl (±)-β-DL38.9 >100 >100 >100 U Carboxamide (±)-β-DL >100 >100 >100 >100 CCarboxamide (±)-β-DL 0.77 >100 >100 >100 >100 C Carboxamide (+)-β-D8.5 >100 >100 >100 C Carboxamide^(b) (−)-β-L 3.6 >100 >100 >100 CN-Methylaminoformyl (±)-β-DL >100 >100 >100 >100 CN,N-Dimethylaminoformyl (±)-β-DL >100 >100 >100 >100 C H (3TC) (−)-β-L0.002 >100 >100 >100 >100 ^(a)Acyclovir used as apositive control had anEC₅₀ of 0.04 μM. ^(b)EC₅₀ against HIV-2_(ROD2) and SIV_(SMM) was 1.6 and4.0 μM, respectively.

EXAMPLE 5 Anti-HBV Activity of 5-Substituted Derivatives of2′,3′-Dideoxy-3′-thiacytidine

The ability of the active compounds to inhibit the growth of virus in2.2.15 cell cultures (HepG2 cells transformed with hepatitis virion) canbe evaluated as described in detail below.

A summary and description of the assay for antiviral effects in thisculture system and the analysis of HBV DNA has been described (Korba andMilman, 1991, Antiviral Res., 15:217). The antiviral evaluations wereperformed on two separate passages of cells. All wells, in all plates,were seeded at the same density and at the same time.

Due to the inherent variations in the levels of both intracellular andextracellular HBV DNA, only depressions greater than 3.5-fold (for HBVvirion DNA) or 3.0-fold (for HBV DNA replication intermediates) from theaverage levels for these HBV DNA forms in untreated cells are consideredto be statistically significant [P<0.05]. The levels of integrated HBVDNA in each cellular DNA preparation (which remain constant on a percell basis in these experiments) were used to calculate the levels ofintracellular HBV DNA forms, thereby ensuring that equal amounts ofcellular DNA were compared between separate samples.

Typical values for extracellular HBV virion DNA in untreated cellsranged from 50 to 150 pg/ml culture medium (average of approximately 76pg/ml). Intracellular HBV DNA replication intermediates in untreatedcells ranged from 50 to 100 pg/μg cell DNA (average approximately 74pg/μg cell DNA). In general, depressions in the levels of intracellularHBV DNA due to treatment with antiviral compounds are less pronounced,and occur more slowly, than depressions in the levels of HBV virion DNA(Korba and Milman, 1991, Antiviral Res., 15:217).

The manner in which the hybridization analyses were performed for theseexperiments resulted in an equivalence of approximately 1.0 pg ofintracellular HBV DNA to 2-3 genomic copies per cell and 1.0 pg/ml ofextracellular HBV DNA to 3×10⁵ viral particles/ml.

Toxicity analyses were performed to assess whether any observedantiviral effects were due to a general effect on cell viability. Themethod used herein was the measurement of the uptake of neutral red dye,a standard and widely used assay for cell viability in a variety ofvirus-host systems, including HSV and HIV. Toxicity analyses wereperformed in 96-well flat bottomed tissue culture plates. Cells for thetoxicity analyses were cultured and treated with test compounds with thesame schedule as described for the antiviral evaluations below. Eachcompound was tested at 4 concentrations, each in triplicate cultures(wells “A”, “B”, and “C”). Uptake of neutral red dye was used todetermine the relative level of toxicity. The absorbance of internalizeddye at 510 nm (A_(sin)) was used for the quantitative analysis.Valuesare presented as a percentage of the average A_(sin) values in 9separate cultures of untreated cells maintained on the same 96-wellplate as the test compounds. Dye uptake in the 9 control cultures onplate 5 ranged from 91.6% to 110.4%, and on plate 6 from 96.6% to 109%.

The results of the HBV assay are provided in Table 2. As indicated, theβ-D and β-L enantiomers of 5-carboxylic acidamide-2′,3′-dideoxy-3′-thiacytidine (referred to as β-L- andβ-D-carboxamide) exhibit significant activity against HBV and arerelatively nontoxic.

TABLE 2 EFFECT OF 5-CARBOXAMIDE DERIVATIVES OF 3TC AGAINST HEPATITIS BVIRUS IN TRANSFECTED HEPG-2 (2.2.15) CELLS ON DAY 9 Selectivity IndexHBV virion^(a) HBV RI^(b) Cytotoxicity IC₅₀/EC₉₀ Compound EC₅₀ ± SD^(c)EC₉₀ ± SD^(c) EC₅₀ ± SD^(c) EC₉₀ ± SD^(c) IC₅₀ ± SD^(c) Virion RIβ-D-DDC 1.4 ± 0.2  9.6 ± 1.1 3.4 ± 0.4 13.0 ± 1.4  236 ± 21  26   18β-L-carboxamide 0.29 ± 0.02  1.5 ± 0.1 1.3 ± 0.1 9.9 ± 0.6 1747 ± 2121165   177 β-D-carboxamide 0.11 ± 0.012 0.9 ± 0.1  0.5 ± 0.04 3.8 ± 0.31124 ± 72  1249   296 β-L-FTC 0.04 ± 0.006 1.1 ± 0.1 0.16 ± 0.01 0.39 ±0.22 746 ± 33  679 1,913 ^(a)Extracellular DNA; untreated control had102 pg/ml ^(b)Replicative intermediates (intracellular DNA), untreatedcontrol had 87 pg/μg cell DNA ^(c)μM

EXAMPLE 5 Anti-HIV Activity of 2′,3′-Didehydro-2′, 3′-dideoxy-pyrimidineNucleosides

Table 3 provides the EC₅₀ values (concentration of nucleoside thatinhibits the replication of the HIV-1 and HIV-2 by 50% in PBM cells,estimated 10% error factor) and IC₅₀ values (concentration of nucleosidethat inhibits 50% of the growth of mitogen-stimulated uninfected humanPBM cells, CEM cells, and in Vero cells) ofβ-L-2′,3′-didehydro-2′,3′-dideoxy-cytidine andβ-L-2′,3′-didehydro-2′,3′-dideoxy-5-fluoro-cytidine. As indicated, bothcompounds exhibit significant activity against HIV, and are relativelynontoxic.

EXAMPLE 5 Anti-HIV Activity of 2′,3′-Didehydro-2′, 3′-dideoxy-pyrimidineNucleosides

TABLE 3 Biological Evaluation of Various β-L-2′,3′-dideoxypyrimidinenucleosides Against HIV-1_(LAI), HIV-2_(ROD2), SIV_(SMM), and forCytotoxicity in PBM, CEM, and Vero Cells. Anti-HIV-1 Anti-HIV-2 Anti-SIVToxicity Toxicity Toxicity in PBMC in PBMC in PBMC in PBM cells in CEMcells in Vero cells Compound Configuation EC₅₀, μM EC₅₀, μM EC₅₀, μMIC₅₀, μM IC₅₀, μM IC₅₀, μM L-D4C (−)-β-L 0.0058 0.033 0.048 >100 0.7310.8 L-F-D4C (−)-β-L 0.0015 0.0006 0.00015 >100 7.3 40.3 3TC (−)-β-L0.002 0.020 0.02 >100 >100 22 100

TABLE 4 Effect of DDC Derivatives Against Hepatitis B Virus (HBV) inTransfected HEpG-2 (2.2.15) Cells on Day 9 Selectivity Index HBVvirion^(a) HBV RI^(b) Cytotoxicity IC₅₀/EC₉₀ Compound EC₅₀ ± SD^(c) EC₉₀± SD^(c) EC₅₀ ± SD^(c) EC₉₀ ± SD^(c) IC₅₀ ± SD^(c) Virion RI β-D-DDC 1.5± 0.2 8.2 ± 0.8 2.4 ± 0.3 12.0 ± 1.1  259 ± 18 37 22 β-L-D4C 0.15 ± 0.020.33 ± 0.04 0.91 ± 0.09 2.3 ± 0.3 1044 ± 92 1149 454 β-L-F-D4C 0.28 ±0.03 0.41 ± 0.04 0.33 ± 0.04 0.75 ± 0.07 >3 >7.3 >4 ^(a)ExtracellularDNA; untreated control had 88 pg/ml ^(b)Replicative intermediates(Intracellular DNA), untreated control had 79 pg/μg cell DNA ^(c)μM

III. Preparation of Pharmaceutical Compositions.

Humans suffering from diseases caused by HIV or HBV infection can betreated by administering to the patient an effective amount of a[5-carboxamido or 5-fluoro]-2′, 3′-dideoxy-2′,3′-didehydro-pyrimidinenucleoside or [5-carboxamido or 5-fluoro]-3′-modified-pyrimidinenucleoside or a pharmaceutically acceptable derivative or salt thereofin the presence of a pharmaceutically acceptable carrier or diluent. Theactive materials can be administered by any appropriate route, forexample, orally, parenterally, intravenously, intradermally,subcutaneously, or topically, in liquid or solid form.

The active compound is included in the pharmaceutically acceptablecarrier or diluent in an amount sufficient to deliver to a patient atherapeutically effective amount of compound to inhibit viralreplication in vivo, especially HIV and HBV replication, without causingserious toxic effects in the patient treated. By “inhibitory amount” ismeant an amount of active ingredient sufficient to exert an inhibitoryeffect as measured by, for example, an assay such as the ones describedherein.

A preferred dose of the compound for all of the above-mentionedconditions will be in the range from about 1 to 50 mg/kg, preferably 1to 20 mg/kg, of body weight per day, more generally 0.1 to about 100 mgper kilogram body weight of the recipient per day. The effective dosagerange of the pharmaceutically acceptable derivatives can be calculatedbased on the weight of the parent nucleoside to be delivered. If thederivative exhibits activity in itself, the effective dosage can beestimated as above using the weight of the derivative, or by other meansknown to those skilled in the art.

The compound is conveniently administered in unit any suitable dosageform, including but not limited to one containing 7 to 3000 mg,preferably 70 to 1400 mg of active ingredient per unit dosage form. Aoral dosage of 50-1000 mg is usually convenient.

Ideally the active ingredient should be administered to achieve peakplasma concentrations of the active compound of from about 0.2 to 70 μM,preferably about 1.0 to 10 μM. This may be achieved, for example, by theintravenous injection of a 0.1 to 5% solution of the active ingredient,optionally in saline, or administered as a bolus of the activeingredient.

The concentration of active compound in the drug composition will dependon absorption, inactivation, and excretion rates of the drug as well asother factors known to those of skill in the art. It is to be noted thatdosage values will also vary with the severity of the condition to bealleviated. It is to be further understood that for any particularsubject, specific dosage regimens should be adjusted over time accordingto the individual need and the professional judgment of the personadministering or supervising the administration of the compositions, andthat the concentration ranges set forth herein are exemplary only andare not intended to limit the scope or practice of the claimedcomposition. The active ingredient may be administered at once, or maybe divided into a number of smaller doses to be administered at varyingintervals of time.

A preferred mode of administration of the active compound is oral. Oralcompositions will generally include an inert diluent or an ediblecarrier. They may be enclosed in gelatin capsules or compressed intotablets. For the purpose of oral therapeutic administration, the activecompound can be incorporated with excipients and used in the form oftablets, troches, or capsules. Pharmaceutically compatible bindingagents, and/or adjuvant materials can be included as part of thecomposition.

The tablets, pills, capsules, troches and the like can contain any ofthe following ingredients, or compounds of a similar nature: a bindersuch as microcrystalline cellulose, gum tragacanth or gelatin; anexcipient such as starch or lactose, a disintegrating agent such asalginic acid, Primogel, or corn starch; a lubricant such as magnesiumstearate or Sterotes; a glidant such as colloidal silicon dioxide; asweetening agent such as sucrose or saccharin; or a flavoring agent suchas peppermint, methyl salicylate, or orange flavoring. When the dosageunit form is a capsule, it can contain, in addition to material of theabove type, a liquid carrier such as a fatty oil. In addition, dosageunit forms can contain various other materials which modify the physicalform of the dosage unit, for example, coatings of sugar, shellac, orother enteric agents.

The compound can be administered as a component of an elixir,suspension, syrup, wafer, chewing gum or the like. A syrup may contain,in addition to the active compounds, sucrose as a sweetening agent andcertain preservatives, dyes and colorings and flavors.

The compound or a pharmaceutically acceptable derivative or saltsthereof can also be mixed with other active materials that do not impairthe desired action, or with materials that supplement the desiredaction, such as antibiotics, antifungals, antiinflammatories, or otherantivirals, including other nucleoside anti-HIV compounds. Solutions orsuspensions used for parenteral, intradermal, subcutaneous, or topicalapplication can include the following components: a sterile diluent suchas water for injection, saline solution, fixed oils, polyethyleneglycols, glycerine, propylene glycol or other synthetic solvents;antibacterial agents such as benzyl alcohol or methyl parabens;antioxidants such as ascorbic acid or sodium bisulfite; chelating agentssuch as ethylenediamrinetetraacetic acid; buffers such as acetates,citrates or phosphates and agents for the adjustment of tonicity such assodium chloride or dextrose. The parental preparation can be enclosed inampoules, disposable syringes or multiple dose vials made of glass orplastic.

If administered intravenously, preferred carriers are physiologicalsaline or phosphate buffered saline (PBS).

In a preferred embodiment, the active compounds are prepared withcarriers that will protect the compound against rapid elimination fromthe body, such as a controlled release formulation, including implantsand microencapsulated delivery systems. Biodegradable, biocompatiblepolymers can be used, such as ethylene vinyl acetate, polyanhydrides,polyglycolic acid, collagen, polyorthoesters, and polylactic acid.Methods for preparation of such formulations will be apparent to thoseskilled in the art. The materials can also be obtained commercially fromAlza Corporation.

Liposomal suspensions (including liposomes targeted to infected cellswith monoclonal antibodies to viral antigens) are also preferred aspharmaceutically acceptable carriers. These may be prepared according tomethods known to those skilled in the art, for example, as described inU.S. Pat. No. 4,522,811 (which is incorporated herein by reference inits entirety). For example, liposome formulations may be prepared bydissolving appropriate lipid(s) (such as stearoyl phosphatidylethanolamine, stearoyl phosphatidyl choline, arachadoyl phosphatidylcholine, and cholesterol) in an inorganic solvent that is thenevaporated, leaving behind a thin film of dried lipid on the surface ofthe container. An aqueous solution of the active compound or itsmonophosphate, diphosphate, and/or triphosphate derivatives is thenintroduced into the container. The container is then swirled by hand tofree lipid material from the sides of the container and to disperselipid aggregates, thereby forming the liposomal suspension.

This invention has been described with reference to its preferredembodiments. Variations and modifications of the invention, will beobvious to those skilled in the art from the foregoing detaileddescription of the invention It is intended that all of these variationsand modifications be included within the scope of the appended claims.

We claim:
 1. A method for treating a host infected with humanimmunodeficiency virus comprising administering an effective amount of aphysiologically acceptable ester ofβ-D-2′,3′-dideoxy-2′,3′-didehydro-5-fluorocytidine (D4FC) or apharmaceutically acceptable salt thereof.
 2. The method of claim 1further comprising administering the ester ofβ-D-2′,3′-dideoxy-2′,3′-didehydro-5-fluorocytidine (D4FC) or apharmaceutically acceptable salt thereof, in combination or alternationwith one or more agents selected from the group consisting of2-hydroxymethyl-5-(5-fluorocytosin-1-yl)-1,3-oxathiolane (FTC),(−)-cis-2′, 3′-dideoxy-3′-thiacytidine (3TC),9-(4-(hydroxymethyl)-2-cyclopenten-1-yl)guanine(carbovir),9-((2-hydroxyethoxy)methyl)guanine(acyclovir), interferon,9-(4-hydroxy-3-(hydroxymethyl)-butyl)guanine(penciclovir),3′-deoxy-3′-azido-thymidine (AZT), 2′,3′-dideoxyinosine (DDI),2′,3′-dideoxycytidine (DDC), (−)-2′-fluoro-5-methyl-β-L-ara-uridine(L-FMAU) and 2′, 3′-didehydro-2′,3′-dideoxythymidine (D4T).
 3. Themethod of claim 1, further comprising administering the ester ofβ-D-2′,3′-dideoxy-2′,3′-didehydro-5-fluorocytidine (D4FC) or apharmaceutically acceptable salt thereof together with apharmaceutically acceptable carrier.
 4. The method of claim 3, whereinthe carrier is suitable for intravenous delivery.
 5. The method of claim3, wherein the carrier is suitable for parenteral delivery.
 6. Themethod of claim 3, wherein the carrier is suitable for intradermaldelivery.
 7. The method of claim 3, wherein the carrier is suitable forsubcutaneous delivery.
 8. The method of claim 3, wherein the carrier issuitable for topical delivery.
 9. The method of claim 1, furthercomprising administering the ester ofβ-D-2′,3′-dideoxy-2′,3′-didehydro-5-fluorocytidine (D4FC) or apharmaceutically acceptable salt thereof in the form of a tablet. 10.The method of claim 3, wherein the carrier is suitable for oraldelivery.